Cytochrome P450 enzymes (CYPs) are critical members of an organism's detoxification systems that help metabolize and eliminate endogenous and exogenous toxic chemicals. The CYPs are phase I enzymes that mono-oxygenate, reduce, and hydrolyze various substrates, yielding more polar, water-soluble metabolites that can be conjugated by phase II enzymes and removed rapidly. The CYPs are also involved in the formation of toxic intermediates and may cause adverse drug reactions (ADRs). It is estimated that between 635,000 and 770,000 patients have a serious adverse drug reaction each year and approximately 106,000 people die from ADRs in the United States (Lazarou et al, J. Am. Med. Assoc. 279:1200 (1998)). This would make ADRs between the 4th and 6th leading cause of death in the United States. Recent research suggests that pharmacogenetic data on CYP polymorphisms will ultimately explain nearly 20% of ADRs. In addition, nearly 50% of ADRs can be explained by physiological or environmental factors, which includes the induction of CYPs (Ingelman-Sundberg et al., J. Inter, Med. 250:186 (2001)).
CYPs are grouped into families, subfamilies and isoforms. The human CYP genes have been arranged into 18 families, 43 subfamilies, and 57 isoforms; the mouse CYP genes have been arranged into 13 families, 43 subfamilies, and 102 isoforms (Nelson et al., Pharmacogenetics 14:1 (2004)). For example, with Cyp3a4, CYP=cytochrome P450, 3=the family, a=the subfamily, and 4 is the isoform. It is the CYPs in families 1-4 that contribute most to the metabolism of xenobiotics, including chemical contaminants and pharmaceuticals (Waxman, Arch Biochem Biophys. 369:11 (1999)). In humans, CYPs such as CYP3A4, CYP2D6, CYP2B6, CYP2C9, and CYP1A2 are very important in xenobiotic metabolism, as well as steroid catabolism, bile acid metabolism, bilirubin elimination, etc. In mice, CYPs such as Cyp3a11, Cyp3a25, Cyp3a41, Cyp2b9, Cyp2b10, Cyp2d9, Cyp2d22, Cyp2c29, Cyp2c37, and Cyp2c40 have similar activities.
The constitutive androstane receptor (CAR) and its cousin, pregnane X receptor (PXR) are relatively new members of the nuclear receptor family that dimerize with retinoid X receptor-α following activation by xenobiotics and endobiotics and in turn act as master regulators of phases I through III enzymes involved in the detoxification and elimination of steroids, bile acids, and xenobiotics. Cyp2b enzymes are inducible by both CAR and PXR, but the Cyp2b enzymes are of special interest with regard to CAR because of the identification of phenobarbital response elements in the 5′ regions of Cyp2b genes and the elucidation of CAR as the receptor that is activated following phenobarbital exposure. Several CAR activators such as phenobarbital, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), phenyloin, nonylphenol, and O-(3,4-dichlorobenzyl)oxime (CITCO) are potent Cyp2b inducers and therefore Cyp2b is an excellent biomarker for CAR activation.
There are more P450 isoforms in each subfamily of the mouse genome than there are in the human genome. For example, in humans there are three members of the Cyp2a subfamily, one member of the Cyp2b subfamily, four members of the Cyp2c subfamily, one member of the Cyp2d subfamily, and four members of the Cyp3a subfamily for a total of 13 isoforms in these subfamilies. In mice, there are four members of the Cyp2a subfamily, five members of the Cyp2b subfamily, 15 members of the Cyp2c subfamily, nine members of the Cyp2d subfamily, and eight members of the Cyp3a subfamily for a total of 41 isoforms in these subfamilies. Table 1 shows the corresponding mouse and human isoforms in each of the principle subfamilies involved in detoxification.
TABLE 1Human and mouse CYP genes involved in detoxification.HumanMouseCYP1A1, 1A2Cyp1a1*, 1a2*CYP1B1Cyp1b1*CYP2A6, 2A7, 2A13Cyp2a4, 2a5, 2a12, 2a22CYP2B6Cyp2b9, 2b10, 2b13, 2b19, 2b23CYP2C8, 2C9, 2C18, 2C19Cyp2c29, 2c37, 2c38, 2c39, 2c40, 2c44, 2c50, 2c54,2c55, 2c65, 2c66, 2c67, 2c68, 2c69, 2c70CYP2D6Cyp2d9, 2d10, 2d11, 2d12, 2d13, 2d22, 2d26, 2d34,2d40CYP2E1Cyp2e1*CYP3A4, 3A5, 3A7, 3A43Cyp3a11, 3a13, 3a16, 3a25, 3a41, 3a44, 3a57, 3a59#CYP4A11, 4A22Cyp4a10, 4a12, 4a14, 4a29, 4a30, 4a31, 4a32CYP4F2, 4F3, 4F8, 4F11, 4F12, 4F22Cyp4f13, 4f14, 4f15, 4f16, 4f17, 4f18, 4f37, 4f39, 4f40*genes for which knockout mice have been produced#The Cyp3a subfamily was recently knocked out by deleting a portion of a chromosome that contained the genes in a tandem repeat region (van Herwaarden et al., J. Clin. Invest. 117: 3583 (2007)).
This redundancy has made typical P450 gene “knockouts” impractical as other subfamily members are available to metabolize a compound of interest. Knocking out Cyp2b10, for example, would have little effect on the physiology of the mouse since Cyp2b9, Cyp2b13, Cyp2b19, and Cyp2b23. are still available to metabolize the compound. Furthermore, the cost of making a pentuplet Cyp2b knockout is prohibitively expensive. Therefore useful knockouts of P450 members have been rare. There are currently six genes knocked out of the 102 mouse members and one subfamily; two involved in bile acid homeostasis (Cyp7a1, Cyp26a1) and four detoxification genes (Cyp1a1, Cyp1a2, Cyp2e1, Cyp1b1, indicated with an asterisk in Table 1 and recently a chromosomal deletion of the CYP3A subfamily where all the members are found in a tandem repeat). This means that there are few P450-null mice for the P450s critical in xenobiotic detoxification. Further, there are almost no P450-null mice for the constitutively expressed xenobiotic detoxifying P450s in the Cyp2-4 families that are also critical in steroid hormone homeostasis, fatty acid metabolism, and bile acid metabolism. Therefore, the exact physiological roles these different CYP families play in vivo in detoxification, steroid hormone homeostasis, and bile acid elimination has not been thoroughly studied using proven, substantiated techniques.
RNA interference (RNAi) technologies have been developed that allow for the “knockdown” of genes. Small interfering RNAs (siRNAs) are short, double-stranded RNA molecules that comprise sequences complementary to mRNAs, and in turn target the mRNA for degradation by hybridizing with the mRNA to form a double stranded RNA (dsRNA) molecule. (Elbashir et al., Genes Dev. 15:188 (2001); Brummelkamp et al., Science 296:550 (2002)). RNAi was first described in Caenorhabditis elegans, when it was discovered that homologous dsRNA resulted in the post-transcriptional silencing of a specific gene (Fire et al., Nature 391:806 (1998)). The gene silencing effect of dsRNA is mediated in a two-step process: 1) the dsRNA is recognized by Dicer, a member of a RNase III family of nucleases, that processes the dsRNA into small double-stranded molecules called siRNAs. 2) The siRNAs are bound by a protein complex called RISC(RNA-induced silencing complex) that contains RNase activity and targets the mRNA for degradation. Typically, siRNA or short hairpin RNA (shRNA) sense and antisense strands are only 21 nucleotides in length because longer dsRNA also elicits an anti-viral interferon response. This results in cessation of all protein synthesis, not just that of the homologous, specific target strand.
The present invention addresses previous shortcomings in the art by providing compositions and methods for the knockdown of multiple members of a P450 subfamily by targeting common mRNA sequences.